Solid-state NMR Spectroscopy detects interactions between tryptophan residues of the E-coli sugar transporter GaIP and the α-anomer of the D-glucose substrate

An experimental approach is described in which high resolution C-13 solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between Specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GaIP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GaIP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers Of D-[1-C-13]glucose were bound by GaIP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the a-anomer of D-[1-C-13]glucose and C-13-labels within [C-13]tryptophanlabeled GalP, which places the carbon atom at C-1 in the a-anomer Of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-C-13]glucose binding to the GaIP Mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.